PROJECT ABSTRACT Approximately 70% of invasive breast cancer expresses estrogen receptor-alpha (ER). Our lab and others have found that 50-60% of early-stage ER+ breast cancers also express glucocorticoid receptor (GR), and that high GR expression provides favorable prognostic information in early-stage breast cancer independently of PR expression. Specifically, in an analysis of primary tumor GR expression in ER+ invasive breast tumors from over 1000 ER+ early-stage patients with >20 year clinical follow-up, we discovered that high tumor GR mRNA expression (and by implication, high GR activity) was associated with a significantly lower risk of relapse. More recently, we found that in ER+ breast cancer cell line models, GR activation remodels chromatin so that ER- chromatin association and ER-target gene expression are significantly altered. Based on these data, we hypothesize that ER and GR can provide coordinated regulation of good prognosis, anti-proliferative and pro- differentiation genes. We propose to determine the molecular mechanisms underlying GR-mediated modulation of both wild-type and mutant ER transcriptional activity, and to define the specific patterns of ER/GR-mediated gene expression in breast cancer. In Aim 1, we will characterize how GR ligand binding domain activation [by either dexamethasone (dex) or novel selective GR modulators (SGRMs)] affects ER activity, consequent ER-mediated gene expression, and ultimately ER-associated tumor cell proliferation. In Aim 2, we will investigate GR and ER chromatin and co-regulator association in the presence of dex in comparison to the novel SGRMs. This will allow us to better understand the requirements of chromatin conformation and GR/ER co-regulator assembly in modifying ER+ breast cancer's proliferative gene expression. Finally, in Aim 3, we will use in vivo ER+ breast cancer patient-derived xenograft models, mutant ER-expressing cell lines, and tamoxifen-resistant ER+ models to define SGRM anti-tumor activity with the aim of defining clinical contexts in which GR modulators are most likely to be effective. Together, these experiments will both increase our understanding of GR/ER crosstalk in breast cancer (and possibly other hormone-sensitive cancers e.g. endometrial) and are expected to lead to new GR-targeted therapies that harness our discovery of anti-proliferative activity following GR modulation in ER+/GR+ breast cancer.